Poliovirus 2C protein determinants of membrane binding and rearrangements in mammalian cells

Abstract

Poliovirus protein 2C is a 329-amino acid-protein that is essential for viral RNA synthesis and may perform multiple functions. In infected cells, it is associated with virus-specific membrane vesicles. Recombinant 2C protein expressed in transfected cells has been shown to associate with and induce rearrangement of the intracellular membrane network. This study was designed to map the determinants of membrane binding and rearrangement in the 2C protein. Computer-assisted analysis of the protein sequence led to a prediction that the protein folds into a structure composed of three domains. Expression plasmids that encode each or combinations of these predicted domains were used to examine the abilities of the partial protein sequences to associate with intracellular membranes and to induce rearrangement of these membranes in HeLa cells. Biochemical fractionation procedures suggested that the N-terminal region of the protein was required for membrane association. Electron microscopic and immunoelectron microscopic observation showed that both the N-and C-terminal regions, but not the central portion, of 2C protein interact with intracellular membranes and induce major changes in their morphology. The central portion, when fused to the N-terminal region, altered the specific membrane architecture induced by the N-terminal region, giving rise to vesicles resembling those observed during poliovirus infection.

Interactions Between Virus Proteins and Host Cell Membranes During the Viral Life Cycle

“…Thus, the 2B moiety in the 2BC protein accounts for transport inhibition (Doedens and Kirkegaard, 1995). The 2BC protein induces vesicles similar to those observed in poliovirus-infected cells, and causes an increase in permeability of the plasma membrane, like 2B (Teterina et al, 1997).…”

“…As opposed to poliovirus 2B, the overexpression of protein 2C does not disrupt glycoprotein traYcking of VSV G protein to the plasma membrane (Suhy et al, 2000). The sequence responsible for membrane binding of 2C has been mapped to its N-terminal region, which has been predicted to fold into an amphipathic a-helix (Echeverri and Dasgupta, 1995;Paul et al, 1994;Teterina et al, 1997). Poliovirus 2C protein displays ATPase (Pfister et al, 2000) and GTPase activities (Rodriguez and Carrasco, 1993) and is involved in genomic replication (Banerjee et al, 1997).…”

Role of host reticulon proteins in rearranging membranes for positive-strand RNA virus replication

“…Although ultrastructural studies were not performed, immunofluorescence showed that endogenous RTN3 colocalized not only with EV71 2C but also with dsRNA, implying that RTN3 is likely associated with the viral replication compartment [38]. The 2C protein is one of the most highly conserved viral proteins among all picornaviruses and poliovirus 2C has been proposed to be involved in the formation of the vesicle-associated replication compartments [39].…”

“…Although ultrastructural studies were not performed, immunofluorescence showed that endogenous RTN3 colocalized not only with EV71 2C but also with dsRNA, implying that RTN3 is likely associated with the viral replication compartment [38]. The 2C protein is one of the most highly conserved viral proteins among all picornaviruses and poliovirus 2C has been proposed to be involved in the formation of the vesicle-associated replication compartments [39].Because of the limitations of conventional, two-dimensional electron microscopy, the RNA replication structures induced by poliovirus and other enteroviruses had been previously described as either single-membrane [40] or double-membrane vesicles (DMVs) [41]. However, EM tomograms of enterovirus-infected cells showed that, during the exponential phase of viral RNA synthesis, closed smooth single-membrane tubules constitute the predominant virus-induced membrane structure, whereas DMVs become increasingly abundant at the expense of these tubules as infection progresses [42 ].…”

Testing the modularity of the N-terminal amphipathic helix conserved in picornavirus 2C proteins and hepatitis C NS5A protein

“…Among the Picornaviridae family members, RNA replication complexes from PV-infected HeLa cells have been the best studied. Viral protein sequences in 2B, 2C and 3A and the larger proteins that contain these sequences all have inherent membrane binding properties (Datta and Dasgupta, 1994;Doedens et al, 1997;Echeverri and Dasgupta, 1995;Teterina et al, 1997b;Towner et al, 1996;van Kuppeveld et al, 1997a). 2B and 2C sequence-containing proteins appear to target to the ER when expressed individually in HeLa cells, although a specific ER-targeting sequence is not known.…”